Many animal and plant pathogenic bacteria, including at least seven on the NIAID priority pathogen list, deliver virulence proteins (effectors) directly into host cells through type III secretion (TTS) systems. There they disrupt cell signaling to manipulate the cell for the bacteria's advantage. This proposal investigates the structural determinants of how specific effector proteins are recognized by the TTS apparatus for subsequent secretion and translocation. Salmonella typhimurium is a class B pathogen that causes severe diarrhea in people and livestock. For some effectors, a small chaperone protein binds to a non-conserved region near the amino-terminus of the effector to ensure efficient translocation. Interestingly, in S. typhimurium, a set of nine effectors have homologous amino-terminal domains (about 145 residues, termed 'WEKIF' domains) but are unrelated elsewhere. These domains are required and sufficient for translocation; no secretion chaperones have been identified and they may not be required. The conserved nature of the domain suggests that it also may localize the effector to specific compartments or proteins in the host cell. The long-term goal of the research is to define the structural basis for the protein:protein interactions involving this domain. This is the first step for discovering potential targets for antimicrobial development. Comparative structural studies of the individual WEKIF domains are proposed here. Aim 1 of the proposed research will improve existing crystals of the WEKIF domain of the SspH1 effector, and begin crystallographic structure determination. Aim 2 proposes to express and purify four other WEKIF domains, SIrP, SifA, Ssel, and SseJ, and screen for crystallization conditions. Following up on a related, but positive result, Aim 3 will screen the full-length SspH1, containing the leucine-rich repeat effector domains, for crystals.